My career goal is to become an independently funded researcher in the field of stem cell biology with expertise in basic mechanisms of ES cell reprogramming and differentiation, especially with respect to blood. The pathway to Independence Award (K99/R00) will greatly assist my transition to full independence by providing funding for both mentored and independent phases. In the immediate future, this award will allow me to gain in depth expertise in stem cell and developmental biology, and hematology, under the mentorship of Dr. George Q. Daley. The training program in the hematology/oncology division at Children's Hospital and Harvard Medical School brings together the resources of both institutions as well as those of the Harvard Stem Cell Institute, and provides an outstanding environment for the completion of training during the mentored phase. This will greatly facilitate my smooth transition to independence. The research described in this proposal is designed to create alternative sources of tissue for hematopoietic transplantation therapies. All patients (e.g. those with childhood leukemia or other genetic disease) unable to receive hematopoietic stem cell transplantation due to lack of suitable donor sources (e.g. matched cord blood, bone marrow, or blood stem cells) stand to benefit. The proof of principle for hematologic disease in the mouse (studies described below) may allow us to expand to transplantation models in other tissues and organs, as well as to patients. The Specific Aims of this proposal are: Specific Aims 1: Generation of histocompatible ctES cells (oocyte free;independent phase) using micro cell mediated chromosome transfer in mouse and human. Specific Aim 2: Testing of hematopoietic potential in ctES cells by in vitro and in vivo methods. The limited availability of human oocytes required for both nuclear transferred (nt) ES and parthenogenetic (p) ES cell techniques (oocyte-mediated methods;mentored phase) poses a significant obstacle to generating customized embryonic stem cells. Therefore, alternative methods to generate similar types of cells are needed. Generation of ES cells by oocyte free methods already has been demonstrated by fusion of somatic cells (sc) and embryonic stem (es) cells to reprogram the somatic cells. However, removal of the genetic material from the resultant tetraploid sc-es hybrid ES cells to make diploid ES cells has not been achieved. As an alternative approach (Specific Aim 1), I propose micro cell-mediated transfer to ES cells of mouse sc-chromosome 17 (human sc-chromosome 6), which contains the major histocompatibility complex (MHC). The resultant duplicated es-chromosome 17 (human es-chromosome 6) will then be removed to make diploid ctES cells in mouse and human. The goal of this research is to create histocompatible patient specific hematopoietic stem cells, which in addition to their potential therapeutic value will provide novel tools for in-depth study of reprogramming and differentiation.